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WO2005115130A2 - Ficus transgénique, procédé de production et utilisation associée - Google Patents

Ficus transgénique, procédé de production et utilisation associée Download PDF

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Publication number
WO2005115130A2
WO2005115130A2 PCT/IL2005/000532 IL2005000532W WO2005115130A2 WO 2005115130 A2 WO2005115130 A2 WO 2005115130A2 IL 2005000532 W IL2005000532 W IL 2005000532W WO 2005115130 A2 WO2005115130 A2 WO 2005115130A2
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WO
WIPO (PCT)
Prior art keywords
plant
ficus
fruit
polynucleotide
protein
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PCT/IL2005/000532
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English (en)
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WO2005115130A3 (fr
Inventor
Moshe Flaishman
Avi Pearl
Sara Golobowicz
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The State Of Israel - Ministry Of Agriculture & Rural Development, Agricultural Research Organization
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Application filed by The State Of Israel - Ministry Of Agriculture & Rural Development, Agricultural Research Organization filed Critical The State Of Israel - Ministry Of Agriculture & Rural Development, Agricultural Research Organization
Priority to US11/597,661 priority Critical patent/US8148603B2/en
Publication of WO2005115130A2 publication Critical patent/WO2005115130A2/fr
Publication of WO2005115130A3 publication Critical patent/WO2005115130A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to genetically modified Ficus plants, particularly
  • Ficus carica fig tree
  • the present invention relates to a method for efficient transformation and regeneration of Ficus carica plants and use thereof for producing trees having improved agricultural traits and for the production of foreign proteins and edible vaccines.
  • Ficus of the Moraceae family is a large genus including about 30 species. Ficus species are commonly used in gardening, in private as well as in public gardens. Most species bear fruit; however, in several species, fruit is considered a problem since the fruit are in-edible and falling fruit litter sidewalks and other pathways.
  • Fig trees are one of the earliest fruit bearing trees cultivated.
  • Ficus carica L. (Moraceae), the well-known fig of commerce is indigenous to areas from Asiatic Turkey to North India, and natural varieties are cultivated in most of the Mediterranean countries.
  • the fig is well known for its nutritive value, and is consumed fresh or as dried fruit worldwide.
  • the fig fruits are also known for their mild laxative activity and high alkalinity, and substances derived therefrom are used in various drug preparations.
  • Other parts of fig trees have also been shown to have a commercial value.
  • US Patent No. 6,235,860 has recently disclosed the use of Ficus carica as a source for natural rubber.
  • 5,494,669 relates to a skin disorder known as pseudofolliculitis barbae (PFB), and more particularly to a preparation for treating PFB.
  • the preparation disclosed is a topical solution comprising a mixture of alophatic alcohol, liquid aloe, liquid camphor, and the soluble materials of the fresh fig leaves of Ficus Carica. Similar compositions are disclosed as a massage composition (US Patent No. 4,582,706).
  • US Patent No. 6,184,193 discloses a shrinkage prevention agent for wet washing of clothing that would conventionally have been dry-cleaned.
  • the shrinkage prevention agent disclosed comprises a steam or vacuum dry-distilled liquid of bark, leaves, stems or flowers of two or more plants, including Ficus carica.
  • Ficus carica Traditional breeding methods of Ficus carica require a long-term effort for improving traits of fig trees.
  • some trees bear only female synconium and others, named caprifig, bear both male and female flowers.
  • True fruits can be produced only on tress bearing female synconium and cross-pollination is mediated by a specific wasp.
  • traits donated by the stamens are hard to track.
  • the application of genetic engineering techniques to stably incorporate homologous and/or heterologous genetic material into woody species, including fruit trees offers the potential of obtaining improved planting stocks for private and public gardens and for agricultural use in a short period of time compared to those developed using traditional breeding techniques.
  • heterologous polypeptides having nutritional and/or pharmaceutical value including edible vaccines.
  • Plants have the capacity to express foreign genes from a wide range of sources, including viral, bacterial, fungal, insect, animal, and other plant species. In single-copy nuclear transgenics, foreign protein in excess of 1% of total protein is often achieved (Hiatt et al., 1989. Nature 342:76-78). Further, assembly and processing of complex animal proteins in plants is possible, e.g., human serum albumin (Sijmons et al., 1990. Bio/Technology 8:217-220) and secretory antibodies (Ma et al., 1995. Science 268:716- 719.).
  • Such vaccines may be based upon bacterial or viral vector systems expressing protective epitopes from diverse pathogens (multivalent vaccines) or may be based upon purified antigens delivered singularly or in combination with relevant antigens or other pathogens.
  • multivalent vaccines multivalent vaccines
  • purified antigens delivered singularly or in combination with relevant antigens or other pathogens include: Several methods for using transgenic plants for oral immunization, for example International Patent Application WO 99/54452; US Patent Nos. 5,484,719; 5,612,487: 5,914,123; 6,034,320; 6,084,152; 6,194,123; 6,395,964 and 6,444,805.
  • the overall efficiency of techniques for genetically modifying plants depends upon the efficiency of the transformation technique(s) used to stably incorporate the required genetic material into plant cells or tissues, and the regeneration technique(s) used to produce viable plants from transformed cells.
  • Ficus religiosa plants have been regenerated from callus of stem segments (Jaiswal andNarayan, 1985. Plant Cell Reports 4:256-258) and leaf callus (Narayan and Jaiswal, 1986.1nd. J. Exper. Biol.24:193-194). Regeneration of Ficus lyrata plants from the axillary buds of a mature tree, and the formation of buds from leaf explants were also reported (Deshpande et al, 1998. Plant Cell Reports 17:571-573). However, for in vitro work with figs, plant regeneration has been restricted to the use of single shoot tips and apical buds.
  • the present invention relates to transgenic Ficus species, particularly to the commercially valuable fig tree Ficus carica, comprising exogenous polynucleotides homologous or heterologous to the target Ficus genome and to transgenic Ficus species capable of expressing exogenous polynucleotides.
  • the present invention further relates to methods for efficient transformation and regeneration of Ficus species.
  • the present invention also relates to genetically modified Ficus species having improved agronomical traits, useful, inter alia, for production of foreign proteins and production of edible vaccines.
  • the present invention provides transgenic Ficus species, specifically Ficus carica, comprising a genetic construct comprising at least one exogenous polynucleotide.
  • the genetic construct further comprises one or more regulatory elements to confer functional expression of the exogenous polynucleotide in the target Ficus plant.
  • regulatory element refers to a non-coding polynucleotide regulating the expression of the exogenous polynucleotide. Regulatory elements include, for example, constitutive, inducible or tissue-specific promoters; enhancer elements; termination elements; transposable elements; and post-transcriptional regulatory elements.
  • the practice of the present invention is not bound to a specific construct and any construct suitable for plant transformation as is known to a person skilled in the art can be used.
  • the genetic construct according to the present invention further comprises a selection marker.
  • Selection markers are well known in the art, and the selection technique may vary depending upon the selection marker used.
  • the selection marker is a gene inducing antibiotic resistance, enabling the survival of the transgenic Ficus plants in a medium containing the antibiotic as a selection agent.
  • the selection marker is a reporter gene.
  • the reporter gene can encode a fluorescent protein, a chemiluminescent protein, a protein having a detectable enzymatic activity and the like, as is known to a person skilled in the art.
  • the selection marker is a herbicide resistance gene, enabling the survival of the transgenic Ficus plants in a medium containing the herbicide as a selection agent.
  • the exogenous polynucleotide is an exogenous polynucleotide selected from the group consisting of polynucleotides homologous to the Ficus genome and polynucleotides heterologous to the Ficus genome.
  • the polynucleotide may be selected from a polynucleotide encoding a polypeptide or a functional portion of a polypeptide, a polynucleotide encoding a regulatory factor, such as a transcription factor, a non-coding polynucleotide such as a regulatory polynucleotide, and antisense polynucleotide that inhibit expression of a specified polypeptide.
  • the transgenic Ficus plant comprises a polynucleotide conferring a desirable agronomical trait selected from the group consisting of, but not limited to, insect tolerance, disease resistance, herbicide tolerance, rooting ability, cold tolerance, drought tolerance, salinity tolerance, modified growth rate, modified fruit development, fruit with better appearance, fruit with better taste, fruit with better storage ability, fruit with better shelf life, and improved dried fruit.
  • the exogenous polynucleotide transformed into the Ficus plant encodes a foreign protein.
  • the term "foreign protein” refers to a polypeptide or a protein not naturally present in the Ficus species to be transformed.
  • the foreign protein is expressed in an edible part of the plant, specifically in the edible fruit of Ficus carica.
  • an edible fig fruit is defined as the multiple fruit developed from the complex fig inflorescence called synconium. The multiple fruit consist of fleshy receptacle surrounding by diminutive drupe that form from each pistillate flower.
  • the foreign protein is expressed in the latex serum produced by the Ficus tree. The foreign protein may be utilized while maintained within the transgenic plant tissue, for example by consumption of transgenic edible Ficus fruit or by employing the latex serum. Alternatively, the foreign protein may be extracted from the transgenic Ficus plants.
  • the Ficus plant is the fig tree Ficus carica and the exogenous polynucleotide encodes a polypeptide contributing to the nutritional composition of the Ficus carica edible fruit.
  • the Ficus plant is the fig tree Ficus carica and the exogenous polynucleotide encodes a protein contributing to the therapeutic value of the Ficus carica edible fruit.
  • the protein is selected from the group consisting of an anti-cancer agent, an anti oxidant and a protein eliciting an immunogenic response in a mammal.
  • the present invention provides a transgenic Ficus carica plant producing fruit comprising at least one exogenous polynucleotide expressing one of the group consisting of a polypeptide, a functional portion of a polypeptide, a peptide, a protein and a fusion protein eliciting immunogenic response in a mammal, wherein the expression is at a level such that upon oral administration to a mammal, an immunogenic response is observed, thus forming an edible vaccine.
  • the vaccine can be any vaccine as is known to a person skilled in the art, including but not limited to a vaccine for the treatment of hepatitis, malaria and cholera.
  • the present invention provides a tissue culture regenerated from the transgenic Ficus plants of the present invention, wherein the tissue culture comprises cells or protoplasts from a transgenic tissue selected from the group consisting of leaves, pollen, embryos, roots, root tips, anthers, flowers, fruit and seeds.
  • the present invention provides a method for producing transgenic Ficus species comprising: a) transforming a genetic construct comprising at least one exogenous polynucleotide into at least one Ficus explant to form a putatively transformed explant; b) inducing the regeneration of at least one adventitious shoot on the putatively transformed explant to obtain putatively transformed adventitious shoot; c) selecting a transformed adventitious shoot; and d) culturing the transformed adventitious shoot to form a transgenic Ficus plantlet.
  • the explants are obtained from an in vitro Ficus culture.
  • the in vitro culture is prepared by horizontally placing shoot tips of Ficus on a solid propagation medium enabling shoot growth and leaf development.
  • the propagation medium comprises at least one cytokinin, at least one auxin and optionally at least one gibberellin.
  • Techniques for transforming genetic constructs into the genome of target plants are well known in the art and include Agrobacterium-mediatQd introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction, and the like. The choice of technique will depend upon the Ficus species to be transformed.
  • Targets for the introduction of the genetic constructs of the present invention include tissues, such as leaf tissues, disseminated cells, protoplasts, seeds, embryos, meristematic regions, cotyledons, hypocotyls, and the like.
  • target plant material for transformation according to the methods of the present invention include explants obtained from in vitro cultures prepared as described above.
  • transformation refers to the introduction of an isolated polynucleotide into a plant cell, either in culture or into the tissues of a plant.
  • transforming the explants is performed by co- culturing the explants with Agrobacterium culture harboring the genetic construct comprising the desired genetic material.
  • the polynucleotide may integrate into the host cell genome ("stable transformation") or be expressed without such integration (“transient transformation”).
  • co-culturing is performed with explants placed on a solid medium, abaxial side up.
  • the term “abaxial” refers to a surface facing away from the plant axis, as the lower side of a leaf; the term “adaxial” refers to a surface facing towards the plant axis, as the upper side of a leaf.
  • transformation is highly dependent on the dorso ventral orientation of the explants. Agrobacterium-mediated transformation occurs only when co-culturing is performed on a solid medium with the explants placed with their abaxial side up.
  • the explants are wounded before culturing with Agrobacterium.
  • the Agrobacterium strain used is Agrobacterium tumefaciens.
  • the co- culturing medium is a solid medium comprising at least one auxin and at least one cytokinin.
  • the Ficus explants taken for transformation are leaf explants. Fully developed leaves are excised from the in vitro shoot culture and taken for transformation. The putatively transformed explants are transferred to conditions inducing the formation of adventitious shoots.
  • the induction of adventitious shoots is obtained by placing the putatively transformed explants, adaxial side up, on a solid regeneration medium under low light intensity; transferring the explants to normal light intensity; and subsequently transferring the explants to a propagation medium under normal light intensity.
  • low light intensity refers to light intensity from about 1 to about 5 ⁇ mol/m s
  • normal light intensity refers to light intensity from about 10 to about 60 ⁇ mol/m s.
  • the regeneration medium comprises at least one auxin, at least one cytokinin and at least one carbon source.
  • the regeneration and the propagation media further comprise at least one selection agent.
  • the selection agent is an antibiotic.
  • the frequency of shoot regeneration obtained by the method of the present invention, for wild type as well as transgenic Ficus species is at least 80%, preferably 90%, more preferably 100%, with regeneration efficacy of at least 3 shoots per explant, preferably 5 shoots per explant, more preferably 10 shoots per explant.
  • the term "regeneration frequency” refers to the percentage of explants forming adventitious shoots.
  • the term “regeneration efficacy” as used herein refers to the number of shoots regenerating on one explant.
  • the explants are leaf explants. After the development of adventitious shoots, only the transformed shoots are selected.
  • the genetic construct of the present invention further comprises a selection marker conferring antibiotic resistance.
  • the transformation method using a selection marker comprises: a) exposing the putatively transformed explants to a regeneration medium comprising a first concentration of a first antibiotic and a second antibiotic, to obtain surviving regenerated shoots; b) transferring the surviving shoots to a propagation medium comprising a second concentration of the first antibiotic and the second antibiotic; c) repeating step (b); and d) transferring the surviving shoots of step (c) to the propagation medium comprising the second antibiotic.
  • the regeneration and propagation media are the media described herein above.
  • the second concentration of the first antibiotic is greater than its first concentration, and the concentration of the second antibiotic is constant.
  • the first antibiotic is kanamycin and the second antibiotic is ticarcillin.
  • the shoots are transferred to a rooting medium and roots are generated using techniques that are well known in the art.
  • Rooted transgenic shoots designated herein as transgenic plantlets, may be grown to mature transgenic Ficus plants.
  • the plantlets include the genetic material introduced using the genetic construct according to the present invention.
  • the products obtained from mature transgenic plants, including fruit, timber, latex serum, wood pulp, fuel wood, and the like, also contain the genetic modification.
  • the transformation and regeneration methods of the present invention provide means for the introduction of at least one exogenous polynucelotide into plants of the genus Ficus, specifically Ficus carica.
  • the polynucleotide can be, for example, new gene, an additional copy of an existent gene, or a regulatory element.
  • the transformation and regeneration methods of the present invention are utilized to introduce genetic material that confers desirable agronomical traits, selected from the group consisting of, but not limited to, insect tolerance, disease resistance, herbicide tolerance, rooting ability, cold tolerance, drought tolerance, salinity tolerance, modified growth rate, modified fruit development, fruit with better appearance, fruit with better taste, fruit with better storage ability, fruit with better shelf life, and improved dried fruit.
  • the genetic material introduced may be homologous or heterologous to the genome of the target plant.
  • the transformation and regeneration methods of the present invention are utilized to introduce into Ficus plants at least one exogenous polynucleotide capable of expressing a foreign protein.
  • Expression of the foreign protein can be constitutive or dependent upon induction, tissue specific or general.
  • the Foreign protein can be extracted from the plant tissue for further purification and use, or can be used while maintained in the Ficus plant tissue.
  • the foreign protein is expressed in an edible part of the plant, specifically in the edible fruit of Ficus carica.
  • the expressed foreign protein contributes to the nutritional composition and value of the fruit.
  • the expressed foreign protein contributes to the therapeutic value of the fruit.
  • the foreign protein is selected from the group consisting of an anti cancer agent, an anti oxidant and a protein eliciting an immunogenic response in a mammal.
  • the transformation and regeneration methods of the present invention are utilized for the production of a transgenic Ficus carica producing fruit comprising protein eliciting immunogenic response in a mammal, wherein the protein is expressed in the plant at a level such that upon oral administration to a mammal, an immunogenic response is observed, thus forming an edible vaccine.
  • the vaccine can be any vaccine as is known to a person skilled in the art, including but not limited to a vaccine for the treatment of hepatitis, malaria and cholera. The present invention is better explained by the description, figures and claims that follow.
  • FIGs. 2a-c show light microscopy observation of shoot organogenesis in the fig tree.
  • Leaf explants of cv. Brown Turkey were cultivated on medium with IBA (2 mg/1) and TDZ (2 mg/1).
  • FIG. 3 shows the influence of sucrose concentration and leaf surface position on adventitious shoot regeneration. Leaf explants of cv. Brown Turkey were cultured on -1 -1 medium with TDZ (2 mg 1 ) and IBA (2 mg 1 ). Vertical bars indicate standard error ( ⁇ S.E.). FIGs.
  • FIGs. 5a-c describe rooting and acclimatization of plants from cv. Brown Turkey.
  • FIG. 6 describes the sensitivity of leaf explants of the fig cvs. Brown Turkey and Smyrna to kanamycin.
  • FIGs. 7a-d show histochemical GUS analysis by stereomicroscope observations.
  • FIGs. 8a-b show PCR analysis of genomic DNA isolated from putatively transgenic shoots of cv. Brown Turkey (A) and Smyrna (B).
  • FIG. 9 presents Southern analysis of genomic DNA isolated from putatively transformed shoot of fig cv. Brown Turkey and Smyrna. The transformed shoots showed the anticipated bands of the pME504 plasmid restriction map.
  • FIG. 10 is a schematic illustration showing a map of the pME504 construct, carrying the nptll, the uidA-intron genes and the bar gene under the control of the CaMV
  • FIG. 11 depict herbicide resistance in control and transgenic Brown Turkey cv treated with increasing concentrations of PPT.
  • FIG. 12 shows transgenic regeneration of 'Brown Turkey' fig cultivar transformed with pME504 and selection on 5 ppm BASTA.
  • the present invention relates to genetically modified plants of the genus Ficus, and parts thereof, transformed with a genetic construct comprising at least one exogenous polynucleotide.
  • the invention further relates to methods of (i) transforming Ficus explants and (ii) regenerating the transformed explants to obtain transgenic Ficus plants.
  • the invention is also relates to products produced by or from the transgenic plants of the present invention.
  • the present invention relates to transgenic fig trees (Ficus carica), and to their use, inter alia, for the production of foreign proteins, as edible vaccines and as improved stocks for agronomical use.
  • the genome of commercially valuable woody plants of the genus Ficus is modified by introducing at least one exogenous polynucleotide, either homologous or heterologous to the Ficus genome.
  • the introduced polynucleotide can modify the production and/or function of a polypeptide of interest, for example increasing the amount of a rate-limiting enzyme by introducing additional copies of the gene.
  • a reduction in the level of a polypeptide of interest can be achieved by transforming the target plant with at least one antisense copy of a gene encoding the polypeptide, or a functional portion thereof.
  • Non-coding portions of polynucleotides such as a regulatory polynucleotide and a polynucleotides encoding regulatory factor such as transcription factor, and/or functional portions of a transcription factor, and/or antisense copy of such a regulatory factor, can also be introduced to the target Ficus plants to modulate the expression of certain polypeptides.
  • An exogenous polypeptide can also be introduced for the production of a foreign protein.
  • the introduced polynucleotide is expressed in a tissue specific manner to produce the foreign protein.
  • the foreign protein is produced in an edible part of the plant, particularly in the fruit of Ficus carica.
  • the fig tree is particularly advantageous as a species for production of foreign proteins due to the prolonged duration of ripening of the fruit on a single tree.
  • Certain fig tree varieties, whether for use as a source of fresh fruit or dried fruit, have been developed to have fruit which are ripening during the course of months, and even the year round. This provides unique advantages for the use of figs as a species suitable for use for fruit-specific production of foreign proteins, and as edible vaccines.
  • Foreign proteins to be produced by the transgenic fig tree are of nutritional and/or pharmaceutical value. As fig fruit are widely consumed, the genetically modified fruit can be used for administering nutritional and/or pharmaceutical compositions to animals and humans. According to yet another embodiment, the foreign protein is expressed in the latex serum produced by Ficus species.
  • the foreign proteins produced by the transgenic fig trees may be isolated from the plant tissue, or may be used while maintained within the plant tissue.
  • the present invention provides transgenic Ficus species comprising a genetic construct comprising at least one exogenous polynucleotide.
  • the Ficus species is Ficus carica.
  • the genetic material includes at least one exogenous polynucleotide desired to be introduced to the target Ficus plant.
  • Genetic constructs introduced into the target plant may comprise a polynucleotide that is homologous and/or heterologous to the target plant genome, and may include polynucleotides encoding a polypeptide or a functional portion of a polypeptide, a polynucleotide encoding a regulatory factor, such as a transcription factor, a non-coding polynucleotides such as regulatory polynucleotides, and antisense polynucleotides that inhibit expression of a specified polypeptide.
  • the genetic construct further comprises at least one regulatory element to confer functional expression of the exogenous polynucleotide in the Ficus plant.
  • polypeptide encompasses amino acid chains of any length, including full-length proteins, wherein amino acid residues are linked by covalent peptide bonds.
  • the genetic construct according to the present invention is built in such a way to further include at least one marker gene conferring the ability to select transformed cells and tissues regenerated therefrom, for example callus, embryos and mature plants.
  • transgenic Ficus plants of the present invention can be transformed with any polynucleotide of interest.
  • polynucleotide means a polymeric collection of nucleotides and includes DNA and corresponding RNA molecules, both sense and anti- sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides.
  • a polynucleotide may be an entire gene, or any portion thereof.
  • Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide” therefore includes all such operable anti-sense fragments.
  • DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a cDNA sequence as a probe to screen an appropriate library.
  • PCR techniques using oligonucleotide primers that are designed based on known genomic DNA, cDNA and protein sequences can be used to amplify and identify genomic and cDNA sequences.
  • Synthetic DNAs corresponding to the identified sequences and variants may be produced by conventional synthetic methods. All of the polynucleotides described herein are isolated and purified, as those terms are commonly used in the art.
  • the genetic construct when the genetic construct comprises a coding portion of a polynucleotide, the genetic construct further comprises a gene promoter sequence and a gene termination sequence operably linked to the polynucleotide to be transcribed.
  • the gene promoter sequence is generally positioned at the 5' end of the polynucleotide to be transcribed, and is employed to initiate transcription of the polynucleotide. Promoter sequences are generally found in the 5' non-coding region of a gene but they may exist in introns or in the coding region. When the construct includes an open reading frame in a sense orientation, the gene promoter sequence also initiates translation of the open reading frame.
  • the gene promoter sequence may comprise a transcription initiation site having an RNA polymerase binding site.
  • the promoter gene sequence, and also the gene termination sequence may be endogenous to the Ficus host or may be exogenous, provided the promoter is functional in species of the genus Ficus.
  • the promoter and termination sequences may be from other plant species, plant viruses, bacterial plasmids and the like. The type of the requested expression will dictate the selection of a suitable promoter.
  • the promoters may be constitutive, inducible, tissue-specific, or developmentally regulated. Promoter hybrids can also be constructed to enhance transcriptional activity (e.g., U.S. Patent No. 5,106,739), or to combine desired transcriptional activity and tissue specificity. Promoters often used for constitutive gene expression in plants include the CaMV 35S promoter, the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter, the mannopine synthase (mas) promoter, the nopaline synthase (nos) promoter, and the octopine synthase (ocs) promoter.
  • a suitable inducible promoter may be selected from genes that are induced during a plant defense response against a parasite infection.
  • a fungal infection triggers an induction of a large number of pathogenesis- related (PR) proteins by the infected plant.
  • PR pathogenesis- related
  • the promoters of these PR sequences may be obtained and utilized in the present invention. Isolation of these PR promoters has been reported from potato plants (e.g., Matton, D.P. and Brisson, N. 1989. Mol Plant Microbe Interact. 2:325-31) and tobacco plants.
  • inducible promoters are heat-shock promoters, a nitrate-inducible promoter derived from the spinach nitrite reductase sequence, hormone-inducible promoters, and light-inducible promoters associated with the small subunit of RuBP carboxylase and light harvesting chloroplast binding protein (LHCP) families.
  • the plant-expressible promoter is a tissue specific promoter. Using tissue specific promoters restricts the expression of the exogenous polynucleotide to the tissue where the promoter is operable.
  • the tissue specific promoter is specific to any one of the tissues forming the edible fig fruit.
  • the gene termination sequence which is located 3' to the DNA sequence to be transcribed, may come from the same gene as the gene promoter sequence or may be the termination sequence of a different gene.
  • Many gene termination sequences known in the art may be usefully employed in the present invention, such as the 3' end of the Agrobacterium tumefaciens nopaline synthase gene.
  • preferred gene terminator sequences are those from the original polypeptide gene, or from the target Ficus species being transformed.
  • the genetic construct of the present invention can further comprise a reporter gene or a selection marker that is effective in the target plant cells to permit the detection of transgenic cells, tissues or plants containing the genetic construct.
  • selection markers and reporter genes typically confer resistance to one or more toxins and encode for a detectable enzymatic activity, respectively.
  • the nptll gene whose expression results in resistance to kanamycin or hygromycin antibiotics, which are generally toxic to plant cells at a moderate concentration, can be used as a selection marker as exemplified herein below.
  • the presence of the desired construct in transgenic cells may be determined by means of other techniques that are well known in the art, including PCR, Southern and Western blots.
  • the transgenic Ficus plant of the present invention comprise a polynucleotide conferring a desirable agronomical trait selected from the group consisting of insect tolerance, disease resistance, herbicide tolerance, rooting ability, cold tolerance, drought tolerance, salinity tolerance, modified growth rate, modified fruit development, fruit with better appearance, fruit with better taste, fruit with better storage ability, fruit with better shelf life, and improved dried fruit.
  • a desirable agronomical trait selected from the group consisting of insect tolerance, disease resistance, herbicide tolerance, rooting ability, cold tolerance, drought tolerance, salinity tolerance, modified growth rate, modified fruit development, fruit with better appearance, fruit with better taste, fruit with better storage ability, fruit with better shelf life, and improved dried fruit.
  • storage ability refers to the time period in which a fresh produce of a plant or parts thereof may be stored before marketing, under favorable conditions, without a significant decrease in its quality.
  • the transgenic Ficus plant comprises a polynucleotide encoding a foreign protein.
  • the term "foreign protein” refers to a polypeptide or a protein not naturally present in the Ficus species to be transformed.
  • the foreign proteins are expressed in an edible part of the plant, specifically in the edible fruit of Ficus carica.
  • foreign proteins can be extracted from the transgenic Ficus plants.
  • the fig is particularly advantageous as a species for production of foreign proteins, specifically for the production of edible vaccines due to the prolonged duration of ripening of the fruit on a single tree, which, in few varieties, is during the year round.
  • the Ficus plant is the fig tree Ficus carica and the exogenous polynucleotide encodes a polypeptide contributing to the nutritional composition of the Ficus carica edible fruit.
  • the Ficus plant is the fig tree Ficus carica and the exogenous polynucleotide encodes a protein contributing to the therapeutic value of the Ficus carica edible fruit.
  • the protein is selected from the group consisting of an anti-cancer agent, an anti oxidant and a protein eliciting an immunogenic response in a mammal.
  • the present invention provides a transgenic Ficus carica plant producing fruit comprising at least one exogenous polynucleotide expressing one of the group consisting of a polypeptide, a functional portion of a polypeptide, a peptide, a protein and a fusion protein eliciting immunogenic response in a mammal, wherein the expression is at a level such that upon oral administration to a mammal, an immunogenic response is observed, thus forming an edible vaccine.
  • Vaccines are administered to human and animal to induce their immune system to produce antibodies against viruses, bacteria and other types of pathogenic microorganisms. Thus, vaccination prevents the occurrence, or reduces severity of a disease once the body is attacked by such pathogens.
  • transgenic plants can be transformed and efficiently express immunogenic protein in an amount sufficient for protein purification and vaccine production as well as for eliciting immunogenic response in a mammal consuming the transgenic plant or parts thereof.
  • Genetic constructs for tissue specific expression are known in the art, thus the immunogenic proteins can be expressed specifically in the plant edible parts. Fig fruit, which are consumed for their nutritional value can therefore provide inexpensive and highly accessible source for various vaccines.
  • the oral vaccines produced by the transgenic plants of the present invention are preferably administered by the consumption of the fruit produced by the transgenic plant.
  • immunogenic compositions derived from the transgenic plant materials suitable for use as more traditional immune vaccines may be also readily prepared from the transgenic plant materials described herein.
  • such immune compositions will comprise a material purified from the transgenic plant.
  • Purification of the antigen may take many forms as is known to those of skill in the art. Whatever initial purification scheme is utilized, the purified material will also be extensively dialyzed to remove undesired small molecular weight molecules (i.e., sugars, pyrogens) and optionally lyophilized for more ready formulation into a desired vehicle.
  • the vaccine can be any vaccine as is known to a person skilled in the art, including but not limited to a vaccine for the treatment of hepatitis, malaria, and cholera.
  • the present invention provides a tissue culture regenerated from the transgenic Ficus plants of the present invention, wherein the tissue culture comprises cells or protoplasts from a tissue selected from the group consisting of leaves, pollen, embryos, roots, root tips, anthers, flowers, fruit and seeds.
  • the present invention provides a method for producing genetically modified Ficus species comprising: a) transforming a genetic construct comprising at least one exogenous polynucleotide into at least one Ficus explant to form a putatively transformed explant; b) inducing the formation of at least one adventitious shoot on the putatively transformed explant to obtain putatively transformed adventitious shoot; c) selecting transformed adventitious shoot; and d) culturing the transformed adventitious shoot to form a transgenic Ficus plantlet.
  • the methods of the present invention employ in vitro fig shoot cultures as a starting material from which the explants are obtained.
  • shoot cultures are known in the art (for example, Pontikis and Melas, 1986. Hort Science 21/1, pp.153). Briefly, cultures are initiates from shoot tips. The elongated shoots are grown on a solid propagation medium until leaves are developed. According to one embodiment, the shoots are placed on a solid propagation medium in a horizontal position in a Magenta box, with multiple shoots per box.
  • the medium comprises at least one auxin, at least one cytokinin and optionally at least one gibberellin.
  • the basal salt mixture used is full strength MS (Murashige and Skoog) medium
  • the auxin is indol-3-butyric acid (IBA)
  • the cytokinin is 6-benzylaminopurine (BA)
  • the gibberellic acid is GA 3 .
  • the medium typically further comprises sucrose as a carbon source.
  • the medium further comprises myoinositol and thiamine-HCl and the pH of the medium is kept in the range of from about 4.5 to about 6.5.
  • the cultures are exposed to a cool fluorescent light in a photoperiod of 16 h of o light and 8 h dark, at 25 C.
  • the light intensity is in the range of between 30 to 2
  • This technology enables the development of transgenic Ficus varieties with improved agronomic performance characteristics, provides a new experimental system for study of gene expression and function in these species, and provides a mean for the production of foreign proteins in edible parts of Ficus species, specifically the production of edible vaccines.
  • the use of Ficus carica varieties improved via the utilization of the transformation and regeneration technology of the present invention also facilitates the implementation of sustainable agricultural practices in fig tree cultivation.
  • Methods for polynucleotide transfer into a plant cell are known in the art, and include, inter alia, Agrobacterium-mediated introduction, protoplast fusion, viral- mediated transformation, high velocity projectile introduction, electroporation, injection into reproductive organs, and injection into immature embryos.
  • genetically modified plants may be obtained by fusion of two distinct protoplasts carrying different genetic material followed by regeneration of the fused protoplast.
  • DNA viruses have been also used as gene vectors in plants.
  • a cauliflower mosaic virus carrying a modified bacterial methotrexate-resistance gene was used to infect a plant, and the foreign gene was systematically spread in the plant.
  • the advantages of this system are the ease of infection, systematic spread within the plant, and multiple copies of the gene per cell.
  • a recently developed procedure for direct gene transfer involves bombardment of cells by microprojectiles carrying DNA (Klein, T. M. et al., 1987. Nature 327:70-72). In this "biolistic" procedure, tungsten or gold particles coated with the exogenous DNA are accelerated toward the target cells.
  • transforming the genetic construct according to the present invention is performed by co-culturing the at least one explant with Agrobacterium harboring the genetic construct comprising the desired exogenous polynucleotide such that the genetic construct is transformed into the explant.
  • Agrobacterium refers to a soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium, which causes crown gall.
  • Agrobacterium includes, but is not limited to, the strains Agrobacterium tumefaciens, (which typically causes crown gall in infected plants).
  • Agrobacterium strains which cause production of nopaline are referred to as "nopaline-type" Agrobacteria; Agrobacterium strains which cause production of octopine (e.g.
  • Agrobacterium strain LBA4404, Ach5, B6) are referred to as "octopine-type" Agrobacteria; and Agrobacterium strains which cause production of agropine (e.g., strain EHA105, EHA101, A281) are referred to as "agropine-type" Agrobacteria.
  • Any A. tumefaciens strain harboring a disarmed Ti plasmid may be used in the methods of the invention utilizing any available Agrobacterium system may be used.
  • Ti plasmid/binary vector system or a co-integrative vector system with one Ti plasmid may be used.
  • A. tumefaciens strain EHA105 is used according to the methods of the present invention.
  • Colonies of. Agrobacterium carrying the genetic construct of interest are prepared for inoculating the explants using methods which are well known to one skilled in the art, as exemplified herein below.
  • Explants taken from in vitro shoot cultures as described above are selected for transformation.
  • the explants are lead explants.
  • the explants are wounded; typically, when leaf explants are used, the leaves are wounded across the midrib.
  • the terms "wounded” or “wounding” as used herein refers to the introduction of a wound in the plant tissue. Wounding of plant tissue may be achieved, for example, by punching, by using a blade, by maceration etc.
  • Inoculation of explants with the Agrobacterium suspension takes place under conditions that optimize infection of the explants.
  • the explants are immersed in the Agrobacterium suspension for at least about twenty minutes, o o at a temperature of about 22 C to 30 C. After incubation, excess suspension is removed and explants are transferred to a solid co-cultivation medium. The explants are placed on the medium with their upper side facing the medium (abaxial side up).
  • the present invention now discloses that transformation is completely dependent on the orientation in which the explants are placed on the medium during co-cultivation, i.e. no transformation occurs when the explants are placed in the opposite orientation (adaxial side up).
  • the co-culturing medium comprises at least one auxin and at least one cytokinin.
  • the auxin is selected from indole-3-butyric acid (IBA) and 2,4-dichlorophenoxy acetic acid (2,4-D) and the cytokinin is thidiazuron (N-phenyl-N'-l,2,3-thiadiazol-5-yl urea, TDZ).
  • co-cultivation is performed for a period of from o o about 65h to about 80h in the dark at a temperature range of 22 C-28 C.
  • Transient transformation refers to the introduction of one or more exogenous polynucleotides into a cell in the absence of integration of the exogenous polynucleotide into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELIS A) which detects the presence of a polypeptide encoded by one or more of the exogenous polynucleotides. Alternatively, transient transformation may be detected by detecting the activity of the protein (e.g.
  • transient transformant refers to a cell which has transiently incorporated one or more exogenous polynucleotides.
  • stable transformation or “stably transformed” refers to the introduction and integration of one or more exogenous polynucleotides into the genome of a cell. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the exogenous polynucleotides.
  • stable transformation of a cell may also be detected by enzyme activity of an integrated gene in growing tissue or by the polymerase chain reaction of genomic DNA of the cell to amplify exogenous polynucleotide sequences.
  • stable transformant refers to a cell which has stably integrated one or more exogenous polynucleotides into the genomic or organellar DNA.
  • the explants removed from the co-cultivation medium are first washed.
  • a preferred washing medium comprises MS medium comprising the anti-bacterial antibiotic ticaracillin, preferably at a concentration of from about 250mg/l to 350mg/l, to eliminate the Agrobacterium.
  • the putatively transformed explants are then transferred to a fresh, solid regeneration medium under conditions which promotes bud regenerating and shoot formation.
  • the regeneration medium further comprises at least one selection agent.
  • the regeneration medium is based on MS medium comprising at least one auxin, at least one cytokinin and at least one carbon source, further comprising at least two selection agents.
  • the present invention discloses a highly efficient system for in vitro regeneration of non-transformed as well as transformed species within the genus Ficus, specifically of fig tree (Ficus carica L.), showing for the first time regeneration frequency of at least 80%, preferably 90%, more preferably 100%.
  • a key factor in the high efficacy of both, transformation and shoot regeneration is the dorsoventral orientation of the explants, specifically leaf explants.
  • the high regeneration frequency is obtained when explants are put on the solid regeneration medium with their adaxial side up. When the explants are put with abaxial side up, although regeneration of shoots is observed, regeneration is apparent only on the free adaxial surface loosing contact with the medium.
  • the difference in the regeneration rate may be due to the morphological differences between the adaxial and abaxial epidermal layers, requiring different threshold levels of hormones for regeneration.
  • a regeneration efficacy of at least 3, preferably 5, more preferably 10 shoots per regenerating explant, is also observed using the methods of the present invention.
  • the auxin in the regeneration medium is IBA at a concentration in the range of 1-3 ppm; the cytokinin is TDZ in a concentration range of
  • the carbon source is sucrose in a concentration range of from about 2% to
  • this combination of growth regulators and carbon source confer optimal shoot regeneration from the transformed leaf explants of Ficus carica.
  • the putatively transformed explants are grown under low light intensity for about one week and are then transferred to normal light intensity. After a growth period of from about 3 weeks to about 10 weeks bud regeneration and adventitious shoot formation is observed. During this growth period, the regenerating shoots are transferred to a propagation medium.
  • the putatively transformed 2 explants are grown under low light intensity of from about 1 to about 5 ⁇ mol/m s and 2 under normal light intensity of from about 10 to about 60 ⁇ mol/m s.
  • the genetic construct of the present invention comprises a selection marker conferring antibiotic resistance.
  • the transformation using a selection marker comprises: a) exposing the putatively transformed explants to a regeneration medium comprising a first concentration of a first antibiotic and a second antibiotic, to obtain surviving regenerated shoots; b) transferring the surviving shoots to a propagation medium comprising a second concentration of the first antibiotic and the second antibiotic; c) repeating step (b); and d) transferring the surviving shoots of step (c) to the propagation medium comprising the second antibiotic.
  • the regeneration and propagation media are the media described above.
  • the second concentration of the first antibiotic is greater than its first concentration, and the concentration of the second antibiotic is constant.
  • the first concentration of the first antibiotic is 50 mg/1
  • the second concentration of the first antibiotic is 100 mg/1
  • the concentration of the second antibiotic is 150 mg/1.
  • the first antibiotic is kanamycin and the second antibiotic is ticarcillin.
  • the root induction medium of the present invention comprises half concentration MS medium supplemented with myo-inositol, thiamine HC1, phloroglucinol, and sucrose, at pH 5.7.
  • the transformed shoots are transferred directly from the regeneration/selection medium to rooting cylinders with soil mixture moistened with root induction medium, wherein the medium optionally comprises 1 mg/1 auxin, preferably IBA.
  • Rooting is accomplished in a period of 10-30 days.
  • plantlets are first kept under high humidity conditions of 50-80% and are then transferred for acclimatization and hardening by a stepwise decrease of the relative humidity.
  • Rooting efficacy (the percentage of transformed shoots which develop healthy root system) according to the present invention is at least 85%, preferably 95%, more preferably 100%.
  • Transformed plantlet may be grown to produce genetically modified mature plants.
  • the transformation and regeneration methods of the present invention are utilized to introduce genetic material that confers desirable agronomical traits, including, but not limited to insect tolerance, disease resistance, herbicide tolerance, rooting ability, cold tolerance, drought tolerance, salinity tolerance, modification of growth rates and properties, fruit with better appearance, fruit with better taste, fruit with better storage ability, fruit with better shelf life, and improved dried fruit.
  • the genetic material introduced may be homologous or heterologous to the genome of the target plant.
  • Introducing new traits to an existing plant variety by traditional breeding methods is a time and work consuming process, even for herbaceous plants and moreover for woody plants having a life cycle of years.
  • using the traditional genetic manipulation is even more complicated, as some trees bear only female synconium and others, named caprifig, bear both male and female flowers.
  • True fruits can be produced only on tress bearing female synconium only.
  • cross-pollination is mediated by a specific wasp. Thus traits donated by the stamens are hard to track and artificial pollination is hard to manipulate.
  • fig Ficus carica, Moraceae
  • the origin of fig is tracked to Western Asia, from where it was spread to the Mediterranean.
  • fig trees are cultivated in many locations with an estimated annual production of one million tons of fruit grown mainly in Turkey and Egypt and also in Morocco, Spain, Greece, California, Italy, Norway, Iran and Tunisia.
  • Approximately 40% of the fig crop is sold as dried fruit, with the remainder dividing between fresh produce, paste, juice and canned preserves.
  • Dried and processed figs not suitable for human consumption can be used as animal fodder.
  • the nutritional value of fresh figs is comparable to that of many other fruits. They are high in calcium.
  • Dried figs with only 20% water are nutritious relative to other fresh fruits
  • There are two main commercial types of figs the "common fig” that produces fruit without pollination, and the "Smyrna fig” that requires pollination by a fig wasp (Blastophaga spp.), that lives in the "caprifig” (male fig), to set fruit.
  • the "common-type” (self-pollinated) fig is more commonly grown. These cultivars bear one or two crops per year. The economic importance of fig production is likely to continue into the future.
  • the short storage and shelf life of fresh fruit, and lack of improved agricultural varieties limits the commercial growth of fig trees.
  • the transformation and regeneration methods of the present invention are utilized to introduce heterologous genetic material for the production of foreign proteins by Ficus carica.
  • the foreign proteins to be produced by such genetically modified plants are proteins that can modulate the nutritional composition and value of the edible fig fruit.
  • the introduced foreign protein is an anti-oxidant.
  • the transformation and regeneration methods of the present invention are utilized to introduce heterologous genetic material for the production of vaccines, particularly to the production of vaccines by the edible fruit of the fig trees.
  • the present invention provides genetically modified Ficus species, specifically Ficus carica plants and parts thereof stably transformed with a genetic construct comprising at least one polynucleotide of interest.
  • the present invention provides a tissue culture regenerated from the genetically modified Ficus plants of the present invention, wherein the tissue culture comprises cells or protoplasts from a tissue selected from the group consisting of leaves, pollen, embryos, roots, root tips, anthers, flowers, fruit and ' seeds.
  • the genetically modified Ficus species are produced by the transformation and regeneration methods of the present invention.
  • the present invention provides a genetically modified Ficus plant wherein the plant is transformed with a genetic construct comprising polynucleotide encoding for at least one protein that is heterologous to the genome of the Ficus plant.
  • the present invention provides Ficus carica plants stably transformed with a genetic construct comprising polynucleotide encoding for at least one polynucleotide, said polynucleotide encodes for a protein contributing to the nutritional value of the Ficus carica edible fruit.
  • the protein is an anti-oxidant.
  • the protein is an anti-cancer agent.
  • the genetically modified plant of the present invention expresses at least one protein that can elicit an immunogenic response in a mammal.
  • the protein eliciting the immunogenic response is expressed in an edible part of the plant, specifically in the fruit.
  • the present invention provides a genetically modified Ficus carica plant producing fruit comprising protein eliciting immunogenic response in a mammal, wherein the protein is expressed in the fruit at a level such that upon oral administration to an animal, an immunogenic response is observed, thus forming an edible vaccine.
  • the transformation and regeneration methods of the present invention can be used to produce any type of vaccine effective in immunizing humans and animals against diseases.
  • Viruses, bacteria, fungi, and parasites that cause diseases in humans and animals can contain antigenic protein(s) which can confer immunity in a human or an animal to the causative pathogen.
  • a DNA sequence coding any of this viral, bacterial, fungal or parasitic antigenic protein may be used in the present invention.
  • Mutant and variant forms of the DNA sequences encoding an antigenic protein which confers immunity to a particular virus, bacteria, fungus or parasite in a mammal (including humans) may also be utilized in this invention.
  • expression vectors may contain DNA coding sequences which are altered so as to change one or more amino acid residues in the antigenic protein expressed in the plant, thereby altering the antigenicity of the expressed protein.
  • Expression vectors containing a DNA sequence encoding only a portion of an antigenic protein as either a smaller peptide or as a component of a new chimeric fusion protein are also included in this invention.
  • the production of the immunogenic protein is directed to the fruit to produce edible vaccine, thus the genetic construct to be used for producing the vaccine comprises a fruit specific promoter, operably linked to a polynucleotide encoding the immunogenic protein.
  • any Agrobacterium delivery system may be employed to transform Ficus carica with the genetic construct.
  • the present invention allows for the production of not only a single vaccine in the Ficus carica fruit but for a plurality of vaccines.
  • Polynucleotides coding for multiple antigenic proteins can be included in the genetic construct, thereby casing the expression of multiple antigenic amino acid sequences in one transgenic plant.
  • a plant may be sequentially or simultaneously transformed with a series of genetic constructs, each of which contains DNA segments encoding one or more antigenic proteins.
  • a transgenic plant expressing multiple antigenic protein sequences can simultaneously elicit an immune response to more than one of these strains, thereby giving disease immunity even though the most prevalent strain is not known in advance.
  • the vaccine produced by the genetically modified Ficus plants according to the present invention can be any vaccine as is known to a person skilled in the art.
  • the vaccine is selected from the group consisting of a vaccine for hepatitis, malaria and cholera.
  • a vaccine for hepatitis for hepatitis, malaria and cholera.
  • the principles of the invention, providing efficient transformation and regeneration methods for the production of genetically modified Ficus carica plants according to the present invention may be better understood with reference to the following non-limiting examples.
  • Fig shoot culture maintenance Fig (Ficus carica L.) cultivars Brown Turkey (common for obtaining fresh produce) and Smyrna (common for dry fruits production) were used.
  • PM proliferation medium
  • Basal salt mixture supplemented with 100 mg/1 myo-inositol, 0.5 mg/1 thiamine-HCl, 3% sucrose (w/v), 0.8% agar (Sigma) and addition of 0.25 mg/1 BA, 0.05 mg/1 IBA and 0.05 mg/1 GA 3 .
  • the pH was adjusted to 5.7 prior to o autoclaving for 20 min. at 120 C. Two shoots were placed horizontally in Magenta boxes, containing 50 ml propagation medium and exposed to cool white fluorescent light 2 o
  • the regeneration media contained MS basal salt mixture, supplemented with 100 mg/1 myo- inositol, 1 mg/1 thiamine-HCl, 2-4% sucrose (w/v) and 0.8%, agar (Sigma), at pH 5.7.
  • the effect of the addition of 0.25% activated charcoal (AC) was also examined.
  • the youngest three expanded leaves isolated from 3-4 weeks old plants were used as explants. Each leaf was wounded forming three scares perpendicular to the central vein and placed on regeneration medium with the abaxial or adaxial surface down. Minimum ten Petri dishes, each containing ten explants were used per treatment.
  • the cultures were 2 kept for 7 days in low light intensity (2.5 ⁇ mol/m s) followed by exposure to high light 2 o intensity (40 ⁇ mol/m s) at 25 C, in a 16hL/8hD photoperiod.
  • Leaf explants were examined after 28 and 35 days and the percentage of explant producing shoots (regeneration capacity) and the mean of adventitious shoots formed per regenerating explant (regeneration efficacy) were recorded. All experiments were repeated at least three times. Rooting and acclimatization Two methods for rooting were examined.
  • RI root induction
  • MS medium half concentration MS medium
  • AC activated charcoal
  • the primers for a 676 bp fragment of the GUS gene were: 17081-001 - GUSup 5' -CGA GCG ATT TGG TCA TGT GAA G- 3' (SEQ D
  • Example 1 Plant regeneration Effect of the growth regulators
  • MS basal medium supplemented with a combination of IBA and TDZ resulted in shoot formation rate of up to 28-30%, while supplementing the MS medium with a combination of NAA and TDZ resulted in a maximum regeneration of only up to 15%.
  • the medium used for the regeneration assays was MS medium comprising different concentrations of IBA and TDZ.
  • Fig. 1A-B shows the effect of growth regulators concentration and two different light conditions on the regeneration of leaf explants obtained from shoot grown in PM medium.
  • leaf explants are isolated from 3-4 weeks old in vitro fig shoot cultures; the explants are placed with their adaxial side up on an MS -based regeneration medium containing 2 mg/l IBA, 2 mg/l TDZ and 4% (w/v) 2 sucrose; explants cultures are exposed to low light intensity (2.5 ⁇ mol/m s) for one week 2 and subsequently transferred to normal light (40 ⁇ mol/m s). Accumulation of the factors that positively influenced regeneration, enable us to develop an efficient and reproducible system for adventitious shoot formation in. This protocol was successfully applied to fig cultivar Brown Turkey and cv.
  • Rooting procedure 1 Two methods were examined to obtained root induction (Table 1). According to the first approach, each regenerated shoot was placed in a tube containing root induction medium comprising 2 mg/l IBA. Using this method, ca. 80% of the shoots formed roots. However, insufficient percentage of these plants was successfully acclimatized in the greenhouse conditions. According to the second approach, regenerated shoots were cultured directly in rooting cylinders, with or without 1 mg/l IBA. After 4 weeks showed 100% root formation was achieved, independent on the presence of an auxin. These plants had better hardening and growth characteristics and easier further acclimatization in the greenhouse conditions (Fig. 5). Table 1: Comparison of Methods for Root Formation
  • Example 2 transformation of Ficus carica Choice of selective antibiotic
  • kanamycin as a selection agent for transformed cells
  • non- inoculated leaf explants from in vitro fig cultures of both cultivars Brown Turkey and Smyrna were tested for their tolerance to kanamycin.
  • Wounded leaves were placed on a regeneration medium supplemented with kanamycin at a concentration selected from 0,
  • explants were initially placed on regeneration medium supplemented with 50 mg/l kanamycin and 150 mg/l ticarcillin and cultured with their adaxial side up. The leaf explants were exposed for one week to low light intensity and then transferred to normal light. Under the selective conditions of 50 mg/l kanamycin, up to 30% of the explants of Brown Turkey and up to 50% of Smyrna formed green shoots after 4 weeks of culture. The regenerating shoots were transferred to PR medium supplemented with 100 mg/l kanamycin and 150 mg/l ticarcillin for further selection. Following this scheme of selection the transformation frequency established for cv.
  • Brown Turkey varied between the experiments from 1.7 to 10 % and from 2.1 to 7.8% for cv. Smyrna.
  • Our observations showed that cultivation of the new emerging transgenic shoots more than 3 subcultures in selective conditions of 100 mg/l kanamycin on proliferation medium resulted in inhibition of their growth and development. Therefore, the following design of regeneration/selection process was concluded: one subculture (4 weeks) on regeneration medium with 50 mg/l kanamycin and 150 mg/l ticarcillin followed by two subcultures (6 weeks) of the isolated shoots on PM with 100 mg/l kanamycin and 150 mg/l ticarcillin and finally one subculture only with ticarcillin (150 mg/l).
  • Example 3 Transgenic ⁇ rown Turkey' plants generated according to the teachings of the present invention expressing the bar herbicide selective marker Bialaphos, a non-selective herbicide, is a tripeptide composed of two L-Ala residues and an analog of Glu known as phosphinothricin (PPT).
  • PPT phosphinothricin
  • Bialaphos is toxic to bacteria and plants after intracellular peptidases remove the Ala residues and release active PPT, an inhibitor of Gin synthetase (GS). Inhibition of GS by PPT causes a rapid buildup of intracellular ammonia levels. The associated disruption of chloroplast structure results in inhibition of photosynthesis and plant cell death. There is thus a widely recognized need for generating plants resistant to PPT, Basta.
  • Transgenic 'Brown Turkey' plants expressing the bar gene were generated using the teachings of the present invention. Such plants are expected to display resistance to the herbicide phosphinothricin (PPT, 'Basta').
  • PPT herbicide phosphinothricin
  • Experimental Procedures and Results The pME504 plasmid which comprises the bar gene [Streptomyces hygroscopicus bar gene conferring resistance to herbicide bialaphos (X05822) Thompson,C.J., Mowa,N.R., Tizard,R., Crameri,R., Davies,J.E., Lauwereys,M. and Botterman,J. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus EMBO J.

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Abstract

L'invention concerne des plantes transgéniques de type Ficus, en particulier Ficus carica (figuier), un procédé de production associé, et des plantes de type Ficus, des matières végétales et des produits végétaux produits par lesdites matières végétales génétiquement modifiées ou obtenues à partir de celles-ci. De manière plus spécifique, l'invention concerne des plantes transgéniques de type Ficus carica et l'utilisation de celles-ci pour la production d'arbres présentant des caractéristiques agricoles améliorées et pour la production de protéines exogènes et de vaccins comestibles.
PCT/IL2005/000532 2004-05-24 2005-05-24 Ficus transgénique, procédé de production et utilisation associée WO2005115130A2 (fr)

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KR100343976B1 (ko) * 1999-05-10 2002-07-22 금호석유화학 주식회사 무화과나무를 이용한 천연고무생산물 및 그 생산방법
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EP2290090A1 (fr) * 2000-08-11 2011-03-02 Syngenta Participations AG Procedes de transformation stable de vegetaux
EP1379659A2 (fr) * 2001-04-06 2004-01-14 Syngenta Participations AG Proteine 80 se liant a une coiffe nucleaire oryza sativa

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111213588A (zh) * 2020-03-11 2020-06-02 钦州市林业科学研究所 一种五指毛桃的简易高效组培繁殖方法
CN111213588B (zh) * 2020-03-11 2022-09-02 钦州市林业科学研究所 一种五指毛桃的简易高效组培繁殖方法

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US8148603B2 (en) 2012-04-03

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